Method for producing recombinant adenovirus

ABSTRACT

The invention concerns a method for producing recombinant adenovirus by which viral DNA is introduced in a packaging cell culture and the viruses produced are harvested after liberation in the supernatant. The invention also concerns the viruses produced and their use.

[0001] The present invention relates to a new process for the productionof recombinant adenoviruses. It also relates to the purified viralpreparations produced according to this process.

[0002] Adenoviruses exhibit certain properties which are particularlyadvantageous for use as vector for the transfer of genes in genetherapy. In particular, they have a fairly broad host spectrum, arecapable of infecting quiescent cells, do not integrate into the genomeof the infected cell, and have not been associated, up until now, withmajor pathologies in man. Adenoviruses have thus been used to transfergenes of interest into the muscle (Ragot et al., Nature 361 (1993) 647),the liver (Jaffe et al., Nature genetics 1 (1992) 372), the nervoussystem (Akli et al., Nature genetics 3 (1993) 224), and the like.

[0003] Adenoviruses are viruses with a linear double-stranded DNA havinga size of about 36 (kilobases) kb. Their genome comprises especially aninverted repeat sequence (ITR) at each end, an encapsidation sequence(Psi), early genes and late genes. The principal early genes arecontained in the E1, E2, E3 and E4 regions. Among these, the genescontained in the El region in particular are necessary for viralpropagation. The principal late genes are contained in the L1 to L5regions. The genome of the Ad5 adenovirus has been completely sequencedand is accessible on a database (see especially Genebank M73260).Likewise, parts or even the whole of other adenoviral genomes (Ad2, Ad7,Ad12 and the like) have also been sequenced.

[0004] For their use in gene therapy, various vectors derived fromadenoviruses have been prepared, incorporating various therapeuticgenes. In each of these constructs, the adenovirus was modified so as torender it incapable of replicating in the infected cell. Thus, theconstructs described in the prior art are adenoviruses from which the Elregion has been deleted, which region is essential for the viralreplication and at the level of which the heterologous DNA sequences areinserted (Levrero et al., Gene 101 (1991) 195; Gosh-Choudhury et al.,Gene 50 (1986) 161). Moreover, to enhance the properties of the vector,it has been proposed to create other deletions or modifications in theadenovirus genome. Thus, a heat-sensitive point mutation was introducedinto the ts125 mutant, making it possible to inactivate the 72 kDa DNAbinding protein (DBP) (Van der Vliet et al., 1975). Other vectorscomprise a deletion of another region essential for the viralreplication and/or propagation, the E4 region. The E4 region is indeedinvolved in the regulation of the expression of the late genes, in thestability of the late nuclear RNAs, in the abolition of the expressionof the host cell proteins and in the efficacy of replication of theviral DNA. Adenoviral vectors in which the E1 and E4 regions are deletedtherefore possess a transcriptional background noise and an expressionof viral genes which are highly reduced. Such vectors have beendescribed for example in Applications WO 94/28152, WO 95/02697,PCT/FR96/00088). In addition, vectors carrying a modification at thelevel of the IVa2 gene have also been described (WO 96/10088).

[0005] The recombinant adenoviruses described in the literature areproduced from different adenovirus serotypes. Indeed, various adenovirusserotypes exist whose structure and properties vary somewhat, but whichexhibit a comparable genetic organization. More particularly, therecombinant adenoviruses may be of human or animal origin. As regardsthe adenoviruses of human origin, there may be mentioned preferablythose classified in group C, in particular the adenoviruses of type 2(Ad2), 5 (Ad5), 7 (Ad7) or 12 (Ad12). Among the various adenoviruses ofanimal origin, there may be mentioned preferably the adenoviruses ofcanine origin, and especially all the strains of the CAV2 adenoviruses[Manhattan strain or A26/61 (ATCC VR-800) for example]. Otheradenoviruses of animal origin are cited especially in application WO94/26914 incorporated into the present by reference.

[0006] In a preferred embodiment of the invention, the recombinantadenovirus is a group C human adenovirus. More preferably, it is an Ad2or Ad5 adenovirus.

[0007] The recombinant adenoviruses are produced in an encapsidationline, that is to say a cell line capable of complementing in trans oneor more functions deficient in the recombinant adenoviral genome. One ofthese lines is for example the line 293 into which a portion of theadenovirus genome has been integrated. More precisely, the line 293 is ahuman embryonic kidney cell line containing the left end (about 11-12%)of the serotype 5 adenovirus (Ad5) genome, comprising the left ITR, theencapsidation region, the E1, including E1a and E1b, region, the regionencoding the pIX protein and a portion of the region encoding the pIVa2protein. This line is capable of transcomplementing recombinantadenoviruses defective for the E1 region, that is to say lacking all orpart of the E1 region, and of producing viral stocks having high titres.This line is also capable of producing, at a permissive temperature (32°C.), virus stocks comprising, in addition, the heat-sensitive E2mutation. Other cell lines capable of complementing the E1 region havebeen described, based especially on human lung carcinoma cells A549 (WO94/28152) or on human retinoblasts (Hum. Gen. Ther. (1996) 215).Moreover, the lines capable of transcomplementing several functions ofthe adenovirus have also been described. In particular, there may bementioned lines complementing the E1 and E4 regions (Yeh et al., J.Virol. 70 (1996) 559; Cancer Gen. Ther. 2 (1995) 322; Krougliak et al.,Hum. Gen. Ther. 6 (1995) 1575) and the lines complementing the E1 and E2regions (WO 94/28152, WO 95/02697, WO 95/27071).

[0008] Recombinant adenoviruses are usually produced by introducingviral DNA into the encapsidation line, followed by lysis of the cellsafter about 2 or 3 days (kinetics of the adenoviral cycle being from 24to 36 hours). After the lysis of the cells, the recombinant viralparticles are isolated by caesium chloride gradient centrifugation.

[0009] For the carrying out of the process, the viral DNA introduced maybe the complete recombinant viral genome, optionally constructed in abacterium (WO 96/25506) or in a yeast (WO 95/03400), transfected intothe cells. It may also be a recombinant virus used to infect theencapsidation line. The viral DNA may also be introduced in the form offragments each carrying a portion of the recombinant viral genome and azone of homology allowing, after introduction into the encapsidationcell, the viral genome to be reconstituted by homologous recombinationbetween the various fragments. A conventional process for the productionof adenoviruses thus comprises the following steps: the cells (forexample the cells 293) are infected in a culture dish with a viralprestock in an amount of from 3 to 5 viral particles per cell(Multiplicity of Infection (MOI) =3 to 5), or transfected with the viralDNA. The incubation then lasts from 40 to 72 hours. The virus is thenreleased from the nucleus by cell lysis, generally by several successivethawing cycles. The cellular lysate obtained is then centrifuged at lowspeed (2000 to 4000 rpm) and the supernatant (clarified cellular lysate)is then purified by centrifugation in the presence of caesium chloridein two steps:

[0010] A first rapid centrifugation of 1.5 hours on two caesium chloridelayers of densities 1.25 and 1.40 flanking the virus density (1.34) soas to separate the virus from the proteins in the medium;

[0011] A second longer gradient centrifugation (from 10 to 40 hoursdepending on the rotor used), which constitutes the actual and solevirus purification step.

[0012] Generally, after the second centrifugation step, the virus bandis predominant. Two fine, less dense bands are nevertheless observedwhose examination by electron microscopy has shown that they are emptyor broken viral particles in the case of the more dense band, and viralsubunits (pentons, hexons) for the less dense band. After this step, thevirus is harvested by piercing, using a needle, in the centrifugationtube and the caesium is removed by dialysis or desalting.

[0013] Although the purity levels obtained are satisfactory, this typeof process has, nevertheless, certain disadvantages. In particular, itis based on the use of caesium chloride, which is a reactive which isincompatible with a therapeutic use in man. Because of this, it isimperative to remove the caesium chloride at the end of thepurification. This process has, in addition, some other disadvantagesmentioned later, which limit its use on an industrial scale.

[0014] To overcome these problems, it has been proposed to purify thevirus obtained after lysis, not using a caesium chloride gradient, butby chromatography. Thus, the article by Huyghe et al. (Hum. Gen. Ther. 6(1996) 1403) describes a study of different types of chromatographiesapplied to the purification of recombinant adenoviruses. This articledescribes especially a study of purification of recombinant adenovirusesusing a weak anion-exchange chromatography (DEAE). Previous studies havealready described the use of this type of chromatography for thispurpose (Klemperer et al., Virology 9 (1959) 536; Philipson L., Virology10 (1960) 459; Haruna et al., Virology 13 (1961) 264). The resultspresented in the article by Huyghe et al. show a fairly mediocreefficacy of the ion-exchange chromatography procedure recommended. Thus,the resolution obtained is average, the authors indicating that virusparticles are present in several chromatography peaks; the yield is low(viral particle yield: 67%; infectious particle yield: 49%); and theviral preparation obtained following this chromatographic step isimpure. In addition, a pretreatment of the virus with variousenzymes/proteins is necessary. This same article describes, moreover, astudy of the use of gel permeation chromatography, demonstrating a verypoor resolution and very low yields (15-20%).

[0015] The present invention describes a new process for the productionof recombinant adenoviruses. The process according to the inventionresults from modifications of the previous processes at the level of theproduction phase and/or at the level of the purification phase. Theprocess according to the invention now makes it possible to obtain veryrapidly and in an industrializable manner virus stocks in a very highquantity and quality.

[0016] One of the first aspects of the invention relates moreparticularly to a process for the preparation of recombinantadenoviruses in which the viruses are harvested from the culturesupernatant. Another aspect of the invention relates to a process forthe preparation of adenoviruses comprising an ultrafiltration step.According to another aspect, the invention also relates to a process forthe purification of recombinant adenoviruses comprising ananion-exchange chromatography step. The present invention also describesan improved purification process using a gel permeation chromatography,optionally coupled to an anion-exchange chromatography. The processaccording to the invention makes it possible to obtain viruses of highquality, in terms of purity, stability, morphology and infectivity, withvery high yields and under production conditions which are completelycompatible with industrial requirements and with the legislationrelating to the production of therapeutic molecules.

[0017] In particular, in terms of industrialization, the process of theinvention uses methods for the treatment of culture supernatants provenon a large scale for recombinant proteins, such as microfiltration ordepth filtration, and tangential ultrafiltration. Moreover, because ofthe stability of the virus at 37° C., this process allows a betterorganization at the industrial stage since, contrary to theintracellular method, the time of harvest does not need to be precise towithin a half-day. Furthermore, it ensures a maximum harvesting of thevirus, which is particularly important in the case of viruses defectivein several regions. Moreover, the process of the invention allows easierand more precise monitoring of the kinetics of production, directly onhomogeneous samples of supernatant, without pretreatment, allowingbetter reproducibility of the productions. The process according to theinvention also makes it possible to dispense with the cell lysis step.The cell lysis has several disadvantages. Thus, it may be difficult toenvisage breaking the cells by freeze-thaw cycles at the industriallevel. Moreover, the alternative methods of lysis (Dounce, X-press,sonication, mechanical shearing, and the like) have disadvantages: theyare potential generators of aerosols which are difficult to confine forthe L2 or L3 viruses (level of confinement of the viruses, dependent ontheir pathogenicity or on their mode of dissemination), these viruseshaving, moreover, a tendency to be infectious by the aerial route; theygenerate shear forces and/or a heat release which are difficult tocontrol, and which reduce the activity of the preparations. The solutionof using detergents to lyse the cells would need to be validated andwould require, in addition, validation of the removal of the detergent.Finally, the cell lysis leads to the presence, in the medium, ofnumerous cell debris which make the purification more complex. In termsof virus quality, the process of the invention potentially allows bettermaturation of the virus, leading to a more homogeneous population. Inparticular, since the packaging of the viral DNA is the last step of theviral cycle, the premature lysis of the cells potentially releases emptyparticles which, although non-replicative, are a priori infectious andcapable of taking part in the toxic effect specific to the virus and ofincreasing the specific activity ratio of the preparations obtained. Thespecific infectivity ratio of a preparation is defined as the ratio ofthe total number of viral particles, measured by biochemical methods(OD260nm, CLHP, PCR, immunoenzymatic methods and the like) to the numberof viral particles generating a biological effect (formation of lysisplaques on cells in culture in solid medium, transduction of cells). Inpractice, for a purified preparation, this ratio is determined bycalculating the ratio of the concentration of the particles measured byOD at 260 nm to the concentration of plaque-forming units of thepreparation. This ratio should be less than 100.

[0018] The results obtained show that the process of the invention makesit possible to obtain a virus of a purity at least equal to itshomologue purified by caesium chloride gradient centrifugation, in asingle step and without prior treatment, starting with a concentratedviral supernatant.

[0019] A first object of the invention therefore relates to a processfor the production of recombinant adenoviruses, characterized in thatthe viral DNA is introduced into a culture of encapsidation cells andthe viruses produced are harvested following release into the culturesupernatant. Contrary to the prior processes in which the viruses areharvested following a premature cell lysis carried out mechanically orchemically, in the process of the invention, the cells are not lysed bymeans of an external factor. The culture is continued for a longerperiod, and the viruses are harvested directly in the supernatant, aftersimultaneous release by the encapsidation cells. The virus according tothe invention is thus recovered in the cell supernatant, whereas in theprior processes, it is an intracellular, more particularly internuclear,virus.

[0020] The Applicant has now shown that, in spite of the extension ofthe duration of the culture and in spite of the use of larger volumes,the process according to the invention makes it possible to generateviral particles in large quantity and of better quality. In addition, asindicated above, this process makes it possible to avoid the lysis stepswhich are cumbersome at the industrial level and generate numerousimpurities.

[0021] The principle of the process is therefore based on the harvestingof the viruses released into the supernatant. This process may involve aculture time greater than that for prior techniques based on the lysisof the cells. As indicated above, the harvesting time does not have tobe precise within a half-day. It is essentially determined by thekinetics of release of the viruses into the culture supernatant.

[0022] The kinetics of release of the viruses may be monitored invarious ways. In particular it is possible to use analytical methodssuch as RP-HPLC, IE-HPLC, semiquantitative PCR (Example 4.3), stainingof dead cells with trypan blue, measurement of the release ofintracellular enzymes of the LDH type, measurement of the particles inthe supernatant by Coulter-type apparatus or by diffraction of light,immunological methods (ELISA, RIA, and the like) or nephelometricmethods, titration by aggregation in the presence of antibodies, and thelike.

[0023] Preferably, the harvesting is carried out when at least 50% ofthe viruses have been released into the supernatant. The time when 50%of the viruses have been released may be easily determined by drawing akinetics according to the methods described above. Still morepreferably, the harvesting is carried out when at least 70% of theviruses have been released into the supernatant. It is particularlypreferable to carry out the harvesting when at least 90% of the viruseshave been released into the supernatant, that is to say when thekinetics reaches a plateau. The kinetics of release of the virus isessentially based on the adenovirus replication cycle and may beinfluenced by certain factors. In particular, it may vary according tothe type of virus used, and especially according to the type of deletionmade in the recombinant viral genome. In particular, the deletion of theE3 region appears to delay the release of the virus. Thus, in thepresence of the E3 region, the virus may be harvested from 24-48 hourspost-infection. In contrast, in the absence of the E3 region, a higherculture time appears to be necessary. In this regard, the Applicant hasperformed experiments on kinetics of release of an adenovirus deficientfor the E1 and E3 regions in the supernatant of the cells, and has shownthat the release starts about 4 to 5 days post-infection, and lasts upto day 14 approximately. The release generally reaches a plateau betweenday 8 and day 14, and the titre remains stable for at least 20 dayspost-infection.

[0024] Preferably, in the process of the invention, the cells arecultured for a period of between 2 and 14 days. Moreover, the release ofthe virus may be induced by expression, in the encapsidation cell, of aprotein, for example a viral protein, involved in the release of thevirus. Thus, in the case of the adenovirus, the release may be modulatedby expression of the Death protein encoded by the E3 region of theadenovirus (protein E3-11.6K), optionally expressed under the control ofan inducible promoter. Because of this, it is possible to reduce thevirus release time and to harvest, in the culture supernatant, more than50% of the viruses 24-48 hours post-infection.

[0025] To recover the viral particles, the culture supernatant isadvantageously filtered beforehand. The adenovirus having a size ofabout 0.1 μm (120 nm), the filtration is performed by means of membraneshaving a porosity sufficiently large to allow the virus to pass through,but sufficiently fine to retain the contaminants. Preferably, thefiltration is carried out by means of membranes having a porositygreater than 0.2 μm. According to a particularly advantageousembodiment, the filtration is carried out by successive filtrations onmembranes of decreasing porosity. Particularly good results wereobtained by carrying out the filtration on depth filters of decreasingporosity 10 μm, 1.0 μm and 0.8-0.2 μm. According to another preferredvariant, the filtration is carried out by tangential microfiltration onflat membranes or hollow fibres. It is possible to use, moreparticularly, flat Millipore membranes or hollow fibres having aporosity of between 0.2 and 0.6 μm. The results presented in theexamples show that this filtration step has a yield of 100% (no loss ofvirus was observed by retention on the filter having the lowestporosity).

[0026] According to another aspect of the invention, the Applicant hasnow developed a process which makes it possible to harvest and purifythe virus from the supernatant. To this effect, the supernatant thusfiltered (or clarified) is subjected to ultrafiltration. Thisultrafiltration makes it possible (i) to concentrate the supernatant,the volumes involved being large, (ii) to carry out a first purificationof the virus and (iii) to adjust the preparation buffer to thesubsequent preparation steps. According to a preferred embodiment, thesupernatant is subjected to tangential ultrafiltration. Tangentialultrafiltration consists in concentrating and fractionating a solutionbetween two compartments, retentate and filtrate, separated by membraneswith a determined cut-off, by creating a flow in the retentatecompartment of the device and by applying a transmembrane pressurebetween this compartment and the filtrate compartment. The flow isgenerally produced by means of a pump in the retentate compartment ofthe device and the transmembrane pressure is controlled by means of avalve on the liquid stream of the retentate circuit or a variablecapacity pump on the liquid stream of the filtrate circuit. The speed offlow and the transmembrane pressure are chosen so as to generate lowshear forces (reynolds number less than 5000 sec⁻¹, preferably less than3000 sec⁻¹, pressure less than 1.0 bar) while avoiding blocking of themembranes. Various systems may be used to carry out the ultrafiltration,such as for example spiral membranes (Millipore, Amicon), flat membranesor hollow fibres (Amicon, Millipore, Sartorius, Pall, GF, Sepracor). Theadenovirus having a mass of about 1000 kDa, membranes having a cut-offof less than 1000 kDa, preferably of between 100 kDa and 1000 kDa, areadvantageously used within the framework of the invention. The use ofmembranes having a cut-off of 1000 kDa or greater indeed causes asubstantial loss of virus at this stage. Preferably, membranes having acut-off of between 200 and 600 kDa, still more preferably between 300and 500 kDa, are used. The experiments presented in the examples showthat the use of a membrane having a cut-off of 300 kDa allows theretention of more than 90% of the viral particles while removingcontaminants from the medium (DNA, proteins in the medium, cellularproteins and the like). The use of a cut-off of 500 kDa offers the sameadvantages.

[0027] The results presented in the Examples show that this step makesit possible to concentrate large volumes of supernatant, without loss ofvirus (yield of 90%), and that it generates a virus of better quality.In particular, concentration factors of 20 to 100 fold can be easilyobtained.

[0028] This ultrafiltration step thus constitutes an additionalpurification compared with the conventional scheme since thecontaminants with a mass less than the cut-off (300 or 500 kDa) areremoved, at least in part. The enhancement of the quality of the viralpreparation is clear when the aspect of the separation is compared afterthe first ultracentrifugation step according to the two processes. Inthe conventional process involving lysis, the tube of viral preparationhas a cloudy appearance with a coagulum (lipids, proteins) whichsometimes touches the virus band, whereas in the process of theinvention, the preparation after release and ultrafiltration has a bandwhich is already well resolved from the contaminants in the medium whichpersist in the top phase. The enhancement of the quality is alsodemonstrated when a comparison is made of the ion-exchangechromatography profiles of a virus obtained by cell lysis relative tothe virus obtained by ultrafiltration as described in the presentinvention. Moreover, it is possible to further enhance the quality bypursuing the ultrafiltration by diafiltration of the concentrate. Thisdiafiltration is carried out on the same principle as the tangentialultrafiltration, and makes it possible to remove more completely thecontaminants of size greater than the membrane cut-off, whileequilibrating the concentrate in the purification buffer.

[0029] Moreover, the Applicant has also shown that this ultrafiltrationthen makes it possible to purify the virus directly by chromatography onan ion-exchange column or by gel permeation chromatography, making itpossible to obtain an excellent resolution of the viral particle peakwithout a need for treating the preparation prior to the chromatography.This is particularly unexpected and advantageous. Indeed, as indicatedin the article by Hyughe et al., cited above, the chromatographicpurification of viral preparations gives mediocre results and furtherrequires a pretreatment of the viral suspension with Benzonase andcyclodextrins.

[0030] More particularly, the process according to the invention istherefore characterized in that the viruses are harvested byultrafiltration.

[0031] As indicated above, the resulting concentrate can be useddirectly for purification of the virus. This purification may be carriedout by previous conventional techniques such as centrifugation on acaesium chloride gradient or another ultracentrifugation medium allowingthe particles to be separated according to their size, density orsedimentation coefficient. The results presented in Example 4 show,indeed, that the virus thus obtained has remarkable characteristics. Inparticular, according to the invention, it is possible to replace thecaesium chloride with a solution of iodixanol,5,5′-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]-bis[N,N′-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide]in which the virus sediments at equilibrium at a relative density ofbetween 1.16 and 1.24. The use of this solution is advantageous because,unlike caesium chloride, it is not toxic. Moreover, the Applicant hasalso shown that, advantageously, the concentrate obtained also makes itpossible to purify the virus directly by an ion-exchange mechanism or bygel permeation, and to obtain an excellent resolution of the viralparticle chromatographic peak without the need for pretreatment.

[0032] According to a preferred embodiment, the viruses are thereforeharvested and purified by anion-exchange chromatography.

[0033] For the anion-exchange chromatography, various types of supportsmay be used, such as cellulose, agarose (Sepharose gels), dextran(Sephadex gels), acrylamide (Sephacryl gels, Trisacryl gels), silica(TSK gels-SW gel), poly[styrene-divinylbenzene] (Source gels or Porosgels), ethyleneglycol-methacrylate copolymer (Toyopearl HW gels and TSKgels-PW gel), or mixtures (agarose-dextran: Superdex gel). Moreover, toenhance the chromatographic resolution, it is preferable, within theframework of the invention, to use supports in the form of beads, havingthe following characteristics:

[0034] as spherical as possible,

[0035] of calibrated diameter (beads which are all identical or whichare as homogeneous as possible), without imperfections or breaks,

[0036] with the smallest possible diameter: beads of 10 μm have beendescribed (MonoBeads from Pharmacia or TSK gel from TosoHaas, forexample). This value appears to constitute the lower limit for thediameter of beads whose porosity should, moreover, be very high in orderto allow penetration of the objects to be chromatographed inside thebeads (see below),

[0037] while remaining rigid in order to withstand pressure.

[0038] Moreover, to chromatograph the adenoviruses which constituteobjects of very large size (diameter >100 nm), it is important to usegels having a high upper limit of porosity, or even as high as possible,so as to allow access of the viral particles to the functional groupswith which they have to interact.

[0039] Advantageously, the support is chosen from agarose, dextran,acrylamide, silica, poly[styrene-divinylbenzene],ethyleneglycol-methacrylate copolymer, alone or as a mixture.

[0040] For the anion-exchange chromatography, the support used should befunctionalized by grafting a group capable of interacting with ananionic molecule. Most generally, the group consists of an amine whichmay be ternary or quaternary. By using a ternary amine, such as DEAE forexample, a weak anion exchanger is obtained. By using a quaternaryamine, a strong anion exchanger is obtained.

[0041] Within the framework of the present invention, it is particularlyadvantageous to use a strong anion exchanger. Thus, a chromatographicsupport as indicated above, functionalized by quaternary amines, ispreferably used according to the invention. Among the supportsfunctionalized by quaternary amines, there may be mentioned, asexamples, the resins Source Q, Mono Q, Q Sepharose, Poros HQ and PorosQE, resins of the Fractogel TMAE type, and the Toyopearl Super Q resins.

[0042] Preferred examples of resins which can be used within theframework of the invention are the Source, especially Source Q, such as15 Q (Pharmacia), the MonoBeads, such as Q (Pharmacia), the Poros HQ andPoros QE type resins. The MonoBeads support (diameter of the beads10±0.5 μm) has been commercially available for more than 10 years andthe resins of the Source (15 μm) or Poros (10 μm or 20 μm) type forabout 5 years. The latter two supports exhibit the advantage of having avery broad internal pore distribution (they range from 20 nm to 1 μm),thus allowing the passage of very large objects through the beads.Furthermore, they offer very little resistance to the circulation ofliquid through the gel (therefore very little pressure) and are veryrigid. The transport of solutes towards the functional groups with whichthey will interact is therefore very rapid. The Applicant has shown thatthese parameters are particularly important in the case of theadenovirus, whose diffusion is slow because of its size.

[0043] The results presented in the Examples show that the adenovirusmay be purified from the concentrate in a single anion-exchangechromatography step, that the purification yield is excellent (140% interms of tdu, compared with the value of 49% reported by Huyghes et al.)and that the resolution is excellent. In addition, the results presentedshow that the adenovirus obtained has a high infectivity, and thereforepossesses the characteristics required for a therapeutic use.Particularly advantageous results were obtained with a strong anionexchanger, that is to say functionalized by quaternary amines, andespecially with the resin Source Q. The resin Source Q15 is particularlypreferred.

[0044] In this regard, another subject of the invention relates to aprocess for the purification of recombinant adenoviruses from abiological medium characterized in that it comprises a step ofpurification by strong anion-exchange chromatography.

[0045] According to this variant, the biological medium may be asupernatant of encapsidation cells producing the said virus, a lysate ofencapsidation cells producing the said virus, or a prepurified solutionof the said virus.

[0046] Preferably, the chromatography is carried out on a supportfunctionalized with a quaternary amine. Still according to a preferredmode, the support is chosen from agarose, dextran, acrylamide, silica,poly[styrene-divinylbenzene], ethyleneglycol-methacrylate copolymer,alone or as a mixture.

[0047] A particularly advantageous embodiment is characterized in thatthe chromatography is performed on a Source Q resin, preferably Q15.

[0048] Moreover, the process described above is advantageously carriedout using a supernatant of producing cells, and comprises a preliminaryultrafiltration step. This step is advantageously carried out under theconditions defined above, and in particular, it is a tangentialultrafiltration on a membrane having a cut-off of between 300 and 500kDa.

[0049] According to another embodiment of the process of the invention,the viruses are harvested and purified by gel permeation chromatography.

[0050] The gel permeation may be performed directly on the supernatant,on the concentrate, or on the virus derived from the anion-exchangechromatography. The supports mentioned for the anion-exchangechromatography may be used in this step, but without functionalization.

[0051] In this regard, the preferred supports are agarose (Sepharosegels), dextran (Sephadex gels), acrylamide (Sephacryl gels, Trisacrylgels), silica (TSK gels-SW gel), ethyleneglycol-methacrylate copolymer(Toyopearl HW gels and TSK gels-PW gel), or mixtures (agarose-dextran:Superdex gel). Particularly preferred supports are:

[0052] Superdex 200HR (Pharmacia)

[0053] Sephacryl S-500HR, S-1000HR or S-2000 (Pharmacia)

[0054] TSK G6000 PW (TosoHaas).

[0055] A preferred process according to the invention thereforecomprises an ultrafiltration followed by an anion-exchangechromatography.

[0056] Another preferred process comprises an ultrafiltration followedby an anion-exchange chromatography, followed by a gel permeationchromatography.

[0057] Another variant of the invention relates to a process for thepurification of adenoviruses from a biological medium comprising a firststep of ultracentrifugation, a second step of dilution or dialysis, anda third step of anion-exchange chromatography. Preferably, according tothis variant, the first step is performed by rapid ultracentrifugationon a caesium chloride gradient. The term rapid means anultracentrifugation ranging from about 0.5 to 4 hours. During the secondstep, the virus is diluted or dialysed against buffer, in order tofacilitate its injection onto the chromatography gel, and theelimination of the ultracentrifugation medium. The third step isperformed using an anion, preferably strong anion, exchangechromatography as described above. In a typical experiment, startingwith the virus harvested in the supernatant (or optionallyintracellular), a 1st rapid ultracentrifugation is performed withcaesium chloride (as in Example 3). Next, after a simple dilution of thesample (for example with 10 volumes of buffer) or after a simpledialysis in buffer, the sample is subjected to ion-exchangechromatography (as in Example 5.1.). The advantage of this variant ofthe process of the invention comes from the fact that it uses 2 totallydifferent modes of separation of the virus (density and surface charge),which may possibly bring the virus to a level of quality combining theperformances of the 2 methods. In addition, the chromatography stepmakes it possible to remove simultaneously the medium used for theultracentrifugation (caesium chloride for example, or any otherequivalent medium mentioned above).

[0058] Another subject of the invention relates to the use of iodixanol,5,5′-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]bis[N,N′-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenedicarboxamide]for the purification of adenoviruses.

[0059] For carrying out the process of the invention, various adenovirusencapsidation cells may be used. In particular, the encapsidation cellsmay be prepared from various pharmaceutically utilizable cells, that isto say cultivatable under industrially acceptable conditions and nothaving a recognized pathogenic character. They may be established celllines or primary cultures and especially human retinoblasts, human lungcarcinoma cells, or embryonic kidney cells. They may be advantageouslycells of human origin which can be infected by an adenovirus. In thisregard, there may be mentioned the KB, Hela, 293, Vero, gmDBP6, HER,A549, HER cells and the like.

[0060] The cells of the KB line are derived from a human epidermalcarcinoma. They are accessible at the ATCC (ref. CCL17) as well as theconditions allowing their culture. The human cell line Hela is derivedfrom a human epithelium carcinoma. It is also accessible at the ATCC(ref. CCL2) as well as the conditions allowing its culture. The line 293cells are human embryonic kidney cells (Graham et al., J. Gen. Virol. 36(1977) 59). This line contains especially, integrated in its genome, theleft part of the genome of the human adenovirus Ad5 (12%). The cell linegm DBP6 (Brough et al., Virology 190 (1992) 624) consists of Hela cellscarrying the adenovirus E2 gene under the control of the MMTV LTR.

[0061] They may also be cells of canine origin (BHK, MDCK, and thelike). In this regard, the cells of the canine line MDCK are preferred.The conditions for the culture of the MDCK cells have been describedespecially by Macatney et al., Science 44 (1988) 9.

[0062] Various encapsidation cell lines have been described in theliterature and are mentioned in the Examples. They are advantageouslycells which transcomplement the adenovirus E1 function. Still morepreferably, they are cells which transcomplement the adenovirus E1 andE4 or E1 and E2a functions. These cells are preferably derived from thehuman embryonic cells of the kidney or the retina, or human lungcarcinomas.

[0063] The invention thus provides a process for the production ofparticularly advantageous recombinant adenoviruses. This process issuited to the production of recombinant viruses which are defective forone or more regions, and in particular of viruses defective for the E1region, or for the E1 and E4 regions. Moreover, it is applicable to theproduction of adenoviruses of various serotypes, as indicated above.

[0064] According to a particularly advantageous mode, the process of theinvention is used for the production of recombinant adenoviruses inwhich the E1 region is inactivated by deletion of a PvuII-BglII fragmentstretching from nucleotide 454 to nucleotide 3328, in the Ad5 adenovirussequence. This sequence is accessible in the literature and also on adatabase (see especially Genebank No. M/3260). In another preferredembodiment, the E1 region is inactivated by deletion of an HinfII-Sau3Afragment stretching from nucleotide 382 to nucleotide 3446. In aspecific mode, the process allows the production of vectors comprising adeletion of the whole of the E4 region. This may be carried out byexcision of an MaeII-MscI fragment corresponding to nucleotides35835-32720. In another specific mode, only a functional part of E4 isdeleted. This part comprises at least the ORF3 and ORF6 frames. By wayof example, these coding frames can be deleted from the genome in theform of PvuII-AluI and BglII-PvuII fragments respectively, correspondingto nucleotides 34801-34329 and 34115-33126 respectively. The deletionsof the E4 region of the virus Ad2 dl808 or of the viruses Ad5 dl1004,Ad5 dl1007, Ad5 dl1011 or Ad5 dl1014 can also be used within theframework of the invention. In this regard, the cells of the inventionare particularly advantageous for the production of viruses comprisingan inactive E1 region and a deletion in the E4 region of the typepresent in the genome of AdS dl1014, that is to say of E4-virusesconserving the reading frame ORF4.

[0065] As indicated above, the deletion in the E1 region coversadvantageously all or part of the E1A and E1B regions. This deletionshould be sufficient to render the virus incapable of autonomousreplication in a cell. The part of the E1 region which is deleted in theadenoviruses according to the invention advantageously coversnucleotides 454-3328 or 382-3446.

[0066] The positions given above refer to the wild-type Ad5 adenovirussequence as published and accessible on a database. Although minorvariations may exist between the various adenovirus serotypes, thesepositions are generally applicable to the construction of recombinantadenoviruses according to the invention from any serotype, andespecially the adenoviruses Ad2 and Ad7.

[0067] Moreover, the adenoviruses produced may possess other alterationsin their genome. In particular, other regions may be deleted in order toincrease the capacity of the virus and reduce its side effects linked tothe expression of viral genes. Thus, all or part of the E3 or IVa2region in particular may be deleted. As regards the E3 region, it mayhowever be particularly advantageous to conserve the part encoding thegp19K protein. This protein indeed makes it possible to prevent theadenoviral vector from becoming the subject of an immune reaction which(i) would limit its action and (ii) could have undesirable side effects.According to a specific mode, the E3 region is deleted and the sequenceencoding the gp19K protein is reintroduced under the control of aheterologous promoter.

[0068] As indicated above, adenoviruses constitute vectors for thetransfer of genes which are very efficient for gene and cell therapyapplications. For that, a heterologous nucleic acid sequence whosetransfer and/or expression into a cell, an organ or an organism isdesired may be inserted into their genome. This sequence may contain oneor more therapeutic genes, such as a gene whose transcription andpossible translation in the target cell generate products having atherapeutic effect. Among the therapeutic products, there may bementioned more particularly enzymes, blood derivatives, hormones,lymphokines: interleukins, interferons, TNF and the like (FR 9203120),growth factors, neurotransmitters or their precursors or synthesisenzymes, trophic factors: BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3,NT5 and the like; apolipoproteins: ApoAI, ApoAIV, ApoE and the like (WO94/25073), dystrophin or a minidystrophin (WO 93/06223), tumoursuppressor genes: p53, Rb, Rap1A, DCC, k-rev and the like (WO 94/24297),genes encoding factors involved in coagulation: factors VII, VIII, IXand the like, suicide genes: thymidine kinase, cytosine deaminase andthe like, or alternatively all or part of a natural or artificialimmunoglobulin (Fab, ScFv and the like, WO 94/29446), and the like. Thetherapeutic gene may also be an antisense gene or sequence, whoseexpression in the target cell makes it possible to control theexpression of genes or the transcription of cellular mRNAs. Suchsequences can for example be transcribed, in the target cell, into RNAswhich are complementary to cellular mRNAs and can thus block theirtranslation into protein, according to the technique described in PatentEP 140 308. The therapeutic gene may also be a gene encoding anantigenic peptide, capable of generating an immune response in man, forthe production of vaccines. They may be especially antigenic peptidesspecific for the Epstein-Barr virus, the HIV virus, the hepatitis Bvirus (EP 185 573), the pseudo-rabies virus, or specific for tumours (EP259 212).

[0069] Generally, the heterologous nucleic acid sequence also comprisesa transcription promoter region which is functional in the infectedcell, as well as a region situated in 3′ of the gene of interest, andwhich specifies a transcriptional end signal and a polyadenylation site.All of these elements constitute the expression cassette. As regards thepromoter region, it may be a promoter region which is naturallyresponsible for the expression of the considered gene when the saidpromoter region is capable of functioning in the infected cell. It mayalso be regions of different origin (which are responsible for theexpression of other proteins, or which are even synthetic). Inparticular, they may be promoter sequences of eukaryotic or viral genesor any promoter or derived sequence, stimulating or repressing thetranscription of a gene in a specific manner or otherwise and in aninducible manner or otherwise. By way of example, they may be promotersequences derived from the genome of the cell which it is desired toinfect, or of the genome of a virus, especially the promoters of theadenovirus MLP, E1A genes, the RSV-LTR, CMV promoter, and the like.Among the eukaryotic promoters, there may also be mentioned theubiquitous promoters (HPRT, vimentin, α-actin, tubulin and the like),the promoters of the intermediate filaments (desmin, neurofilaments,keratin, GFAP and the like), the promoters of therapeutic genes (MDR,CFTR, factor VIII type and the like), the tissue-specific promoters(pyruvate kinase, villin, the promoter for the intestinal fattyacid-binding protein, the promoter for α-actin of the smooth musclecells, promoters specific for the liver; Apo AI, Apo AII, human albuminand the like) or alternatively the promoters which respond to a stimulus(steroid hormone receptor, retinoic acid receptor and the like). Inaddition these expression sequences may be modified by the addition ofactivating or regulatory sequences or of sequences allowing atissue-specific or predominant expression. Moreover, when the insertednucleic acid does not contain expression sequences, it may be insertedinto the genome of the defective virus downstream of such a sequence.

[0070] Moreover, the heterologous nucleic acid sequence may alsocontain, in particular upstream of the therapeutic gene, a signalsequence directing the synthesized therapeutic product in the secretorypathways of the target cell. This signal sequence may be the naturalsignal sequence for the therapeutic product, but it may also be anyother functional signal sequence or an artificial signal sequence.

[0071] The expression cassette for the therapeutic gene may be insertedinto various sites of the genome of the recombinant adenovirus,according to the techniques described in the prior art. It can first ofall be inserted at the level of the E1 deletion. It can also be insertedat the level of the E3 region, as an addition or as a substitution ofsequences. It can also be located at the level of the deleted E4 region.

[0072] The present invention also relates to the purified viralpreparations obtained according to the process of the invention, as wellas any pharmaceutical composition comprising one or more defectiverecombinant adenoviruses prepared according to this process. Thepharmaceutical compositions of the invention can be formulated for atopical, oral, parenteral, intranasal, intravenous, intramuscular,subcutaneous, intraocular or transdermal administration and the like.

[0073] Preferably, the pharmaceutical composition contains vehicleswhich are pharmaceutically acceptable for an injectable formulation.These may be in particular saline (monosodium or disodium phosphate,sodium, potassium, calcium or magnesium chloride and the like, ormixtures of such salts), sterile or isotonic solutions, or dry,especially freeze-dried, compositions which, upon addition depending onthe case of sterilized water or physiological saline, allow theconstitution of injectable solutions. Other excipients can be used, suchas, for example a hydrogel. This hydrogel can be prepared from anybiocompatible and noncytotoxic polymer (homo or hetero). Such polymershave for example been described in Application WO 93/08845. Some ofthem, such as especially those obtained from ethylene and/or propyleneoxide, are commercially available. The virus doses used for theinjection can be adjusted according to various parameters, andespecially according to the mode of administration used, the relevantpathology, the gene to be expressed, or the desired duration oftreatment. In general, the recombinant adenoviruses according to theinvention are formulated and administered in the form of doses ofbetween 10⁴ and 10¹⁴ pfu, and preferably 10⁶ to 10¹⁰ pfu. The term pfu(plaque forming unit) corresponds to the infectivity of an adenovirussolution, and is determined by infecting an appropriate cell culture andmeasuring, generally after 15 days, the number of infected cell plaques.The techniques for determining the pfu titre of a viral solution arewell documented in the literature.

[0074] Depending on the therapeutic gene, the viruses thus produced canbe used for the treatment or the prevention of numerous pathologies,including genetic diseases (dystrophy, cystic fibrosis and the like),neurodegenerative diseases (Alzheimer, Parkinson, ALS and the like),cancers, pathologies linked to coagulation disorders or todyslipoproteinaemias, pathologies linked to viral infections (hepatitis,AIDS and the like), and the like.

[0075] The present invention will be more fully described with the aidof the following examples which should be considered as illustrative andnonlimiting.

[0076] Legend to the figures

[0077]FIG. 1: Study of the stability of the adenovirus purifiedaccording to Example 4.

[0078]FIG. 2: HPLC (reversed phase) analysis of the adenovirus purifiedaccording to Example 4. Comparison with the adenovirus of Example 3.

[0079]FIG. 3: Kinetics of release of the adenovirus Ad-βGal in thesupernatant of cells 293, measured by semiquantitative PCR and PlaqueAssay.

[0080]FIG. 4: Elution profile on Source Q15 of an ultrafilteredadenovirus supernatant (Example 5.1).

[0081]FIG. 5: Ressource Q HPLC analysis of the virus peak harvested bychromatography on a Source Q15 resin of an ultrafiltered adenovirussupernatant (Example 5.1).

[0082]FIG. 6: (A) Elution profile on a Source Q15 resin of anultrafiltered Ad-APOA1 adenovirus supernatant (Example 5.3); and (B)HPLC (Resource Q) analysis of the virus peak harvested.

[0083]FIG. 7: (A) Elution profile on Source Q15 of an ultrafilteredAd-TK adenovirus supernatant (Example 5.3). HPLC (Resource Q) analysisof the various virus fractions harvested (start and end of peak): (B)Fraction F2, middle of the peak; (C) Fraction F3, limit of the peak; (D)Fraction F4, end of the peak.

[0084]FIG. 8: Elution profile on a Mono Q resin of a concentratedsupernatant of culture of adenovirus producing cells (Example 5.4).BG25F1: Supernatant virus concentrated and purified on caesium. BG25C:Concentrated infected supernatant.

[0085]FIG. 9: Elution profile on a POROS HQ gel of a concentratedsupernatant of culture of adenovirus producing cells (Example 5.4).BG25F1: Virus supernatant concentrated and purified on caesium. BG25C:Concentrated infected supernatant.

[0086] FIGS. 10 A and B: Gel permeation purification profile onSephacryl S1000HR/Superdex 200HR of an ultrafiltered adenovirussupernatant (Example 6).

[0087]FIG. 11: Analysis by electron microscopy of a stock of adenoviruspurified according to the invention.

[0088]FIG. 12: Analysis by electron microscopy of the virus band ofdensity 1.27.

GENERAL MOLECULAR BIOLOGY TECHNIQUES

[0089] The methods conventionally used in molecular biology, such aspreparative extractions of plasmid DNA, centrifugation of plasmid DNA incaesium chloride gradient, agarose or acrylamide gel electrophoresis,purification of DNA fragments by electroelution, phenol orphenol-chloroform extraction of proteins, ethanol or isopropanolprecipitation of DNA in saline medium, transformation in Escherichiacoli and the like, are well known to persons skilled in the art and arewidely described in the literature [Maniatis T. et al., “MolecularCloning, a Laboratory Manual”, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y., 1982; Ausubel F.M. et al., (eds), “CurrentProtocols in Molecular Biology”, John Wiley & Sons, New York, 19871].

[0090] The pBR322 and pUC type plasmids and the phages of the M13 seriesare of commercial origin (Bethesda Research Laboratories). For theligations, the DNA fragments can be separated according to their size byagarose or acrylamide gel electrophoresis, extracted with phenol or witha phenol/chloroform mixture, precipitated with ethanol and thenincubated in the presence of phage T4 DNA ligase (Biolabs) according tothe recommendations of the supplier. The filling of the protruding 5′ends can be performed with the Klenow fragment of E. coli DNA polymeraseI (Biolabs) according to the specifications of the supplier. Thedestruction of the protruding 3′ ends is performed in the presence ofphage T4 DNA polymerase (Biolabs) used according to the recommendationsof the manufacturer. The destruction of the protruding 5′ ends isperformed by a controlled treatment with S1 nuclease.

[0091] Site-directed mutagenesis in vitro by syntheticoligodeoxynucleotides can be performed according to the method describedby Taylor et al., [Nucleic Acids Res. 13 (1985) 8749-8764] using the kitdistributed by Amersham. The enzymatic amplification of the DNAfragments by the so-called PCR technique [Polymerase-catalyzed ChainReaction, Saiki R. K. et al., Science 230 (1985) 1350-1354; Mullis K. B.and Faloona F. A., Meth. Enzym. 155 (1987) 335-350] can be performedusing a DNA thermal cycler (Perkin Elmer Cetus) according to thespecifications of the manufacturer. The verification of the nucleotidesequences can be performed by the method developed by Sanger et al.,[Proc. Natl. Acad. Sci. USA, 74 (1977) 5463-5467] using the kitdistributed by Amersham.

EXAMPLES Example 1 Encapsidation Cell Lines

[0092] The encapsidation cells used within the framework of theinvention may be obtained from any cell line which can be infected by anadenovirus and which is compatible with a use for therapeutic purposes.They are more preferably a cell chosen from the following lines:

[0093] The cells of the 293 line:

[0094] The 293 line is a human embryonic kidney cell line containing theleft end (about 11-12%) of the genome of the serotype 5 adenovirus(Ad5), comprising the left ITR, the encapsidation region, the E1 region,including E1a, E1b, the region encoding the pIX protein and a portion ofthe region encoding the pIVa2 protein (Graham et al., J. Gen. Virol. 36(1977) 59). This line is capable of transcomplementing recombinantadenoviruses defective for the E1 region, that is to say lacking all orpart of the E1 region, and of producing viral stocks having high titres.

[0095] The cells of the A549 line

[0096] Cells complementing the adenovirus E1 region were constructedfrom the A549 cells (Imler et al., Gene Ther. (1996) 75). These cellscontain a restricted fragment of the E1 region, lacking the left ITR,placed under the control of an inducible promoter.

[0097] The cells of the HER line

[0098] The human embryonic retinal (HER) cells may be infected with anadenovirus (Byrd et al., Oncogene 2 (1988) 477). Adenovirusencapsidation cells prepared from these cells have been described forexample in Application WO 94/28152 or in the article by Fallaux et al.(Hum. Gene Ther. (1996) 215). There may be mentioned more particularlythe line 911 comprising the E1 region of the Ad5 adenovirus genome, fromnucleotide 79 to nucleotide 5789 integrated into the genome of HERcells. This cell line allows the production of viruses defective for theE1 region.

[0099] The IGRP2 cells

[0100] The IGRP2 cells are cells obtained from cells 293, by integrationof a functional unit of the E4 region under the control of an induciblepromoter. These cells allow the production of viruses defective for theE1 and E4 regions (Yeh et al., J. Virol (1996) 70).

[0101] The VK cells

[0102] The VK cells (VK2-20 and VK10-9) are cells obtained from cells293, by integration of the entire E4 region under the control of aninducible promoter, and of the region encoding the pIX protein. Thesecells allow the production of viruses defective for the E1 and E4regions (Krougliak et al., Hum. Gene Ther. 6 (1995) 1575).

[0103] The 293E4 cells

[0104] The 293E4 cells are cells obtained from cells 293, by integrationof the entire E4 region. These cells allow the production of virusesdefective for the E1 and E4 regions (WO 95/02697; Cancer Gene Ther.(1995) 322).

Example 2 Viruses Used

[0105] The viruses produced in the context of the examples which followare an adenovirus containing the E. coli LacZ marker gene (Ad-βGal), anadenovirus containing the gene encoding the type I herpes virusthymidine kinase (Ad-TK), an adenovirus containing the gene encoding thehuman p53 tumour suppressor protein and a virus encoding theapolipoprotein A1 (Ad-apoAI). These viruses are derived from the Ad5serotype and possess the following structure:

[0106] A deletion in the E1 region covering, for example, nucleotides382 (HinfI site) to 3446 (Sau3a site).

[0107] A cassette for expression of the gene, under the control of theRSV or CMV promoter, inserted at the level of the said deletion.

[0108] A deletion of the E3 region.

[0109] The construction of these viruses has been described in theliterature (WO 94/25073, WO 95/14102, FR 95.01632, Stratford-Perricaudetet al. J. Clin. Invest (1992) p626). It is understood that any otherconstruct may be produced according to the process of the invention, andespecially viruses carrying other heterologous genes and/or otherdeletions (E1/E4 or E1/E2 for example).

Example 3 Production of Virus by Lysis of the Cells

[0110] This example reproduces the previous technique for producingviruses, consisting in lysing the encapsidation cells in order torecover the viruses produced.

[0111] The cells 293 are infected at 80-90% confluence in a culture dishwith a prestock of Ad-βGal or Ad-TK virus (Example 2) in an amount of 3to 5 viruses per cell (Multiplicity of Infection MOI =3 to 5). Theincubation lasts for 40 to 72 hours, the timing of the harvest beingdetermined by observing, under a microscope, cells which become round,become more refringent and adhere increasingly weakly to the culturesupport. In the literature, the kinetics of the viral cycle lasts for 24to 36 hours.

[0112] At the level of the laboratory production, it is important toharvest the cells before they become detached so as to remove theinfection medium at the time of the harvest without losing cells andthen to take them up in a minimum volume (the concentration factor is,depending on the size of the culture, of the order of 10 to 100 fold).

[0113] The virus is then released from the nucleus by 3 to 6 successivethawing cycles (ethanol-dry ice at −70° C., water bath at 37° C.).

[0114] The cell lysate obtained is then centrifuged at low speed (2000to 4000 rpm) and the supernatant (clarified cell lysate) is thenpurified by ultracentrifugation on a caesium chloride gradient in twosteps:

[0115] A first rapid ultracentrifugation (step) of 1.5 hours, 35000 rpm,rotor sw 41, on two caesium layers 1.25 and 1.40 flanking the virusdensity (1.34) so as to separate the virus from the proteins in themedium; the rotors may be “swinging” rotors (Sw28, Sw41 Beckman) orfixed angle rotors (Ti 45, Ti 70, Ti 70.1 Beckman) depending on thevolumes to be treated;

[0116] A second, longer gradient ultracentrifugation (from 10 to 40hours depending on the rotor used), for example 18 hours at 35000 rpm insw 41 rotor which constitutes the actual and sole virus purification.The virus is present in a linear gradient at equilibrium at a density of1.34.

[0117] Generally, at this stage, the virus band is predominant.Nevertheless, two fine, less dense bands are sometimes observed whoseexamination by electron microscopy has shown that they are empty orbroken viruses and for the least dense band, viral subunits (pentons,hexons). After this step, the virus is harvested in the tube by piercingwith a needle and the caesium is removed by dialysis or desalting onG25.

Example 4 Production of Virus in the Supernatant

[0118] This example describes an experiment for the production of virusby recovering following spontaneous release. The virus is then harvestedby ultrafiltration and then purified by caesium chloride.

[0119] 4.1. Procedure

[0120] In this method, unlike Example 3, the cells are not harvested 40to 72 hours post-infection, but the incubation is extended between 8 to12 days so as to obtain total lysis of the cells without the need tocarry out the freeze-thaw cycles. The virus is present in thesupernatant.

[0121] The supernatant is then clarified by filtration on depth filtersof decreasing porosity (10 μm/1.0 μm/0.8-0.2 μm).

[0122] The virus has a size of 0.1 μm and at this stage, no loss ofvirus by retention on the filter at the lowest porosity (0.22 μm) wasobserved.

[0123] The supernatant, once clarified, is then concentrated bytangential ultrafiltration on a Millipore spiral membrane having acut-off of 300 kDa.

[0124] In the experiments reported in the present invention, theconcentration factor is dictated by the dead volume of the system whichis 100 ml. Supernatant volumes of 4 to 20 liters were concentrated withthis system, making it possible to obtain concentrate volumes of 100 mlto 200 ml without difficulty, which corresponds to a concentrationfactor of 20 to 100 fold.

[0125] The concentrate is then filtered on 0.22 μm and then purified bycentrifugation on caesium chloride as described in Example 3, followedby a dialysis step.

[0126] 4.2 Results

[0127] Purity

[0128] Whereas the intracellular virus tube (Example 3) has a cloudyappearance with a coagulum (lipids, proteins) which sometimes touchesthe virus band, the viral preparation obtained after the firstcentrifugation step on caesium chloride by the process of the inventionhas a virus band which is already well isolated from the contaminants inthe medium which persist in the top phase. High-performance liquidchromatography analysis on a Resource Q column (cf Example 5) also showsthis gain in the purity of the starting material obtained byultrafiltration of infected supernatant with a decrease in the nucleicacid contaminants (OD 260/280 ratio greater than or equal to 1.8) andprotein contaminants (OD 260/280 ratio less than 1).

[0129] Stability of the Virus in a Supernatant at 37° C.

[0130] The stability of the virus was determined by titration, by theplaque assay method, of an infectious culture supernatant of whichaliquots were collected at various incubation times at 37° C.post-infection. The results are presented below:

[0131] Ad-TK virus:

[0132] titre 10 days post-infection=3.5×10⁸ pfu/ml

[0133] titre 20 days post-infection=3.3×10⁸ pfu/ml Ad-βGal virus

[0134] titre 8 days post-infection=5.8×10⁸ pfu/ml

[0135] titre 9 days post-infection=3.6×10⁸ pfu/ml

[0136] titre 10 days post-infection=3.5×10⁸ pfu/ml

[0137] titre 13 days post-infection=4.1×10⁸ pfu/ml

[0138] titre 16 days post-infection=5.5×10⁸ pfu/ml

[0139] The results obtained show that up to at least 20 dayspost-infection, the titre of the supernatant is stable within the limitsof precision of the assay. Moreover, FIG. 1 shows that, in the elutionbuffer, the virus is stable for at least 8 months, at −80° C. and at−20° C.

[0140] Specific Infectivity of the Preparations

[0141] This parameter, corresponding to the ratio of the number of viralparticles measured by HPLC to the number of pfu, gives information onthe infectivity of the viral preparations. According to therecommendations of the FDA, it should be less than 100. This parameterwas measured as follows: Two series of culture flasks containing cells293 were infected in parallel at the same time with the same viralprestock under the same conditions. This experiment was carried out fora recombinant Ad-βgal adenovirus, and then repeated for an Ad-TKadenovirus. For each adenovirus, a series of flasks is harvested 48hours post-infection and is considered as a production of intracellularvirus purified on a caesium gradient after freezing-thawing. The otherseries is incubated for 10 days post-infection and the virus isharvested in the supernatant. The purified preparations obtained aretitrated by plaque assay and the quantification of the total number ofviral particles is determined by measuring the concentration of PVIIprotein by reversed phase HPLC on a C4 Vydac 4.6×50 mm column afterdenaturation of the samples in 6.4 M guanidine. The quantity of PVIIproteins is correlated to the number of viral particles considering 720copies of PVII per virus (Horwitz, Virology, second edition (1990)).This method is correlated with the measurements of viral particles onpreparations purified by the densitometric method at 260 nm, taking asspecific extinction coefficient 1.0 unit of absorbance =1.1×10¹²particles per ml.

[0142] The results obtained show that, for the Ad-βGal virus, this ratiois 16 for the supernatant virus and 45 for the intracellular virus. Forthe Ad-TK virus, the ratio is 78 in the case of the virus harvest in thesupernatant method and 80 for the virus harvested by the intracellularmethod.

[0143] Analysis by Electron Microscopy

[0144] This method makes it possible to detect the presence of emptyparticles or free viral subunits copurified, and to assess a proteincontamination of the purified viral preparations or the presence ofnon-dissociable aggregates of viral particles.

[0145] Procedure: 20 μl of sample are deposited on a carbon grid andthen treated for the examination by negative staining with 1.5% uranylacetate. For the examination, a 50 kV to 100 kV Jeol 1010 electronmicroscope is used.

[0146] Result: The analysis carried out on a virus harvested in thesupernatant shows a clean preparation, without contaminants, withoutaggregates and without empty viral particles. It is furthermore possibleto distinguish the virus fibres as well as its regular geometricstructure. These results confirm the high quality of the viral particlesobtained according to the invention.

[0147] HPLC and SDS-PAGE Analysis of the Protein Profile

[0148] SDS-PAGE Analysis

[0149] 20 μl of sample is diluted in Laemmli buffer (Nature 227 (1970)680-685), reduced for 5 min at 95° C., and then loaded onto Novex gels 1mm×10 wells gradient 4-20%. After migration, the gels are stained withcoomassie blue, and analysed on a Pharmacia VDS Image Master. Theanalysis reveals an electrophoretic profile for the virus harvested inthe supernatant in agreement with the literature data (LennartPhilipson, 1984 H. S. Ginsberg editors, Plenum press, 310-338).

[0150] Reversed phase HPLC analysis

[0151]FIG. 2 shows the superposition of 3 chromatograms obtained fromtwo virus samples harvested intracellularly and a virus sample purifiedby the supernatant method. The experimental conditions are thefollowing: Vydac column ref. 254 Tp 5405, RPC4 4.6×50 mm, Solvent\A:H20+TFA 0.07%; Solvent B:CH3CN+TFA 0.07%, linear gradient: T=0 min%B=25; T-50 min %B=50%; flow rate=1 ml/min, detector=215 nm. Thechromatograms show a perfect identity between the samples, withoutdifference in the relative intensities of each peak. The nature of eachpeak was determined by sequencing and shows that the proteins presentare all of viral origin (see table below). PEAK (Min) IDENTIFICATION19-20 Precursor PVII 21-22 Precursor PVII; Precursor PX 1 to 12 27-28Precursor PVI; Precursor PX 32-33 Precursor PX 34 35-36 Mature PVII 37Mature PVII; PVIII precursor 39-41 Mature PVI 45 pX 46 pIX

[0152] Analysis in Vitro of the Efficiency of Transduction and of theCytotoxicity

[0153] The analysis of the cytotoxicity is carried out by infectingHCT116 cells in 24-well plates for increasing MOIs and by determiningthe percentage of live cells compared with a non-infected control, 2 and5 days post-infection, with the aid of the crystal violet stainingtechnique.

[0154] The results are presented in the table below: Adenovirus MOI =3.0 MOI = 10.0 MOI = 30.0 MOI = 100.0 Supernatant, D2 91% 96% 87% 89%Supernatant, D5 97% 90% 10% <5%

[0155] Analysis of the transduction efficiency

[0156] For an AD-βGal adenovirus, the transduction efficiency of apreparation is determined by infecting W162 cells, non-permissive toreplication, cultured in 24-well plates, with increasing concentrationsof viral particles. For the same quantity of viral particles deposited,the cells expressing the beta-galactosidase activity are counted 48hours post-infection after incubation with X-gal as substrate. Each bluecell is counted as one transduction unit (TDU), the result is multipliedby the dilution of the sample so as to obtain the concentration in unitsof transduction of the sample. The transduction efficiency is thenexpressed by calculating the ratio of the concentration of viralparticles to the concentration in TDU. The results obtained show thatthe purified viruses have a good transduction efficiency in vitro.

[0157] Analysis of the Intracerebral Expression in Vivo

[0158] With the aim of evaluating the efficiency of the adenovirusesaccording to the invention for the transfer and expression of genes invivo, the adenoviruses were injected by the stereotaxic route into thestriatum of OF1 immunocompetent mice. For that, volumes of 1 μl at 10⁷pfu of virus were injected at the following stereotaxic reference points(for the incision line at 0 mm): anteroposterior: +0.5; mediolateral: 2;depth: −3.5.

[0159] The brains were analysed 7 days after the injection. The resultsobtained show that the transduction efficiency is high: thousands oftransduced cells, very intense expression in the nucleus and frequentand intense diffusion in the cytoplasm.

[0160] 4.3. Kinetics of Release of the Virus

[0161] This example describes a study of the kinetics of release ofadenoviruses in the encapsidation cell culture supernatant.

[0162] This study was carried out by semiquantitative PCR by means ofoligonucleotides complementary to the regions of the adenovirus genome.To this effect, the linearized viral DNA (1-10 ng) was incubated in thepresence of dXTP (2 μl, 10 mM), a pair of specific oligonucleotides andTaq polymerase (Cetus) in a 10X PCR buffer, and subjected to 30amplification cycles under the following conditions: 2 min at 91° C., 30cycles (1 min 91° C., 2 min at annealing temperature, 3 min at 72° C.),5 min 72° C., then 4° C. PCR experiments were carried out with the pairsof oligonucleotides of sequence: Pair 1: TAATTACCTGGGCGGCGAGCACGAT(6368) - SEQ ID No. 1 ACCTTGGATGGGACCGCTGGGAACA (6369) - SEQ ID No. 2Pair 2: TTTTTGATGCGTTTCTTACCTCTGG (6362) - SEQ ID No. 3CAGACAGCGATGCGGAAGAGAGTGA (6363) - SEQ ID No. 4 Pair 3:TGTTCCCAGCGGTCCCATCCAAGGT (6364) - SEQ ID No. 5AAGGACAAGCAGCCGAAGTAGAAGA (6365) - SEQ ID No. 6 Pair 4:GGATGATATGGTTGGACGCTGGAAG (6366) - SEQ ID No. 7AGGGCGGATGCGACGACACTGACTT (6367) - SEQ ID No. 8

[0163] The quantity of free adenovirus in the supernatant was determinedon a supernatant of cells 293 infected with Ad-βGal, at various timespost-infection. The results obtained are presented in FIG. 3. They showthat the cellular release starts from the 5th or 6th day post-infection.

[0164] It is understood that any other virus determination technique maybe used with the same objective, on any other encapsidation line, andfor any adenovirus type.

Example 5 Purification of the Virus by Ultrafiltration and Ion Exchange

[0165] This example illustrates how the adenovirus contained in theconcentrate may be purified directly and in a single ion-exchangechromatography step, with very high yields.

[0166] 5.1. Procedure

[0167] In this experiment, the starting material therefore consists ofthe concentrate (or ultrafiltration retentate) described in Example 4.This retentate has a total protein content of between 5 and 50 mg/ml,and more preferably between 10 and 30 mg/ml, in PBS buffer (10 mMphosphate, pH 7.2 containing 150 mM NaCl).

[0168] The ultrafiltration supernatant obtained from a virus preparationis injected into a column containing Source Q 15 (Pharmacia)equilibrated in 50 mM Tris-HCl buffer pH 8.0 containing 250 mM NaCl, 1.0mM MgCl₂ and 10% glycerol (buffer A). After rinsing with 10 columnvolumes of buffer A, the adsorbed species are eluted with a linear NaClgradient (250 mM to 1 M) on 25 column volumes at a linear flow rate of60 to 300 cm/h, more preferably 12 cm/h. The typical elution profileobtained at 260 nm is presented in FIG. 4. The fraction containing theviral particles is collected. It corresponds to a fine symmetrical peakwhose retention time coincides with the retention time obtained with apreparation of viral particles purified by ultracentrifugation. It ispossible to inject, under the conditions described above, a minimum of30 mg of total proteins per ml of Source Q 15 resin while preserving anexcellent resolution of the viral particle peak.

[0169] In a representative experiment carried out using a β-galadenovirus preparation (Example 2), 12.6 mg of total proteins wereinjected onto a Resource Q column (1 ml), that is to say 5×10¹⁰ PFU and1.6×10¹⁰ TDU. The viral particle peak collected after chromatography(3.2 ml; FIG. 5) contained 173 μg of proteins and 3.2×10¹⁰ PFU and2.3×10¹⁰ TDU. The viral particles were therefore purified 70 fold (interms of quantity of proteins) and the purification yield is 64% in PFUand 142% in TDU (see table below). Concentration Volumes VolumesPurification Steps sample deposited recovered Yields factor SUPERNATANT5000 ml 5000 ml 100% — ULTRA- Proteins: 6.3 mg/ml 5000 ml  200 ml 100% 5 FILTRATION PFU: 2.5 × 10¹⁰/ml 300 kd TDU: 8.1 × 10⁹/ml Particle 3.8 ×10¹¹/ml Part/pfu ratio: 16.0 Part/tdu ratio: 47.0 ION-EXCHANGE PFU: 1.0× 10¹⁰/ml 2.0 ml of 3.0 ml Proteins = 85% PURIFICATION TDU: 7.2 × 10⁹/mlconcentrate of elution PFU = 64% 70 (one step) Particle: 2.0 × 10¹¹/mlTDU = 140% Part/pfu ratio = 20 Particles = 84% Part/tdu ratio = 27 HPLCPurity = 98.4% CsCl PFU: 1.0 × 10¹¹/ml 28.3 ml of QSP 4.1 ml PFU = 66%GRADIENT TDU: 7.5 × 10¹⁰/ml concentrate TDU = 130% 70 Particle: 2.2 ×10¹²/ml Particles = 84% Part/pfu ratio = 22 HPLC Part/tdu ratio = 29purity = 98.4%

[0170] 5.2. Purity

[0171] After this purification step, the fraction collected has a purity≧98% of viral particles (UV detection at 260 nm), when it is analysed byhigh-performance liquid chromatography (HPLC) on a Resource Q column (1ml) in the following chromatographic system: (10 μl of fraction purifiedby chromatography as described in Example 5.1 are injected into aResource Q15 column (1 ml of gel; Pharmacia) equilibrated in 50 mMTris/HCl buffer pH 8.0 (buffer B). After rinsing with 5 ml of buffer B,the adsorbed species are eluted with a linear gradient of 30 ml of NaCl(0 to 1 M) in the B buffer at a flow rate of 1 ml/min. The elutedspecies are detected at 260 nm. This HPLC analysis (FIG. 5) shows,furthermore, that the residual bovine serum albumin present in theultrafiltration retentate is completely removed during the preparativechromatography. Its content in the purified fraction is estimated to be<0.1%. Western blot analysis with an anti-BSA polyclonal antibody (withECL revealing; Amersham) indicates that the content of BSA in thechromatographic preparation is less than 100 ng per mg of virus.

[0172] The electrophoretic analysis of the adenoviral fraction purifiedby chromatography is performed on a polyacrylamide gel (4-20%) underdenaturing (SDS) conditions. The protein bands are then revealed withsilver nitrate. This analysis shows that the adenoviral preparationobtained by chromatography has a purity level at least equal to that ofthe preparation conventionally obtained by ultracentrifugation since ithas no additional protein band which would indicate a contamination ofthe preparation by non-adenoviral proteins.

[0173] The adenoviral preparation obtained by chromatography has anabsorbance ratio A_(260 nm)/A_(280 nm) equal to 1.30±0.05. This value,which is identical to that obtained for the best preparations obtainedby ultracentrifugation, indicates that the preparation is free ofcontaminating proteins or of contaminating nucleic acids.

[0174] Analysis by electron microscopy carried out under the conditionsdescribed in Example 4.2 on a chromatography-purified Ad-βgal virusshows a clean preparation, without contaminants, without aggregates andwithout empty viral particles (FIG. 11). Furthermore, caesium chloridegradient ultracentrifugation of this preparation reveals a single bandof density 1.30, which confirms the absence of contamination of thechromatographic preparations by potential empty particles or capsidfragments. During the purification, the chromatographic peak for thevirus is followed by a shoulder (or secondary peak) in its rear portion,which is not collected with the main peak. Caesium chloride gradientultracentrifugation of this fraction reveals a band of density 1.27, andanalysis of the composition of this fraction shows that it does notcontain nucleic acids. Analysis by electron microscopy shows that thisfraction contains particles of irregular shape, exhibiting perforationsat the surface (FIG. 12). They are therefore empty (lacking DNA) andincomplete particles. This therefore demonstrates that the purificationof the adenovirus by chromatography eliminates the empty particlespresent in a small quantity in the preparations before purification.

[0175] 5.3. Purification of Adenoviruses Comprising a Therapeutic Genesuch as the Genes Encoding the ApoA1 or Thymidine Kinase Proteins.

[0176] This example illustrates how adenoviruses comprising, in theirgenomes, heterologous nucleic acid sequences encoding therapeuticproteins may be purified directly and in a single ion-exchangechromatography step. It also shows that the chromatographic behaviour ofthe adenovirus is identical to the heterologous nucleic acid sequenceswhich it carries, allowing the same purification process to be used fordifferent adenoviruses carrying various heterologous nucleic acidsequences.

[0177] In a typical purification experiment, an adenovirus comprising,in its genome, a heterologous nucleic acid sequence encoding the ApoA1protein (Example 2, WO 94/25073) is purified by chromatographing, in thesystem described in Example 5.1, 18 ml (72 mg of proteins; 1.08×10¹³particles) of concentrated supernatant of a cell culture harvested 10days post-infection (FIG. 6A). The viral particle peak collected afterchromatography (14 ml; 1.4 mg of proteins) contained 9.98×10¹²particles, which indicates a particle yield of 92% and a purificationfactor of 51. After this purification step, the fraction collected had(FIG. 6B) a purity greater than 98% as viral particles during thechromatographic analysis under the conditions described in 5.2. Theelectrophoretic analysis of the adenoviral fraction purified bychromatography carried out under the conditions described in Example 5.2showed that this preparation had a purity level at least equal to thatfor the preparation conventionally obtained by ultracentrifugation, andthat it is free of contaminating proteins or of contaminating nucleicacids.

[0178] In another typical purification experiment, an adenoviruscomprising, in its genome, a heterologous nucleic acid sequence encodingthe type 1 herpes simplex thymidine kinase protein (Example 2, WO95/14102) is purified by chromatographing, in the system described inExample 5.1, 36 ml (180 mg of proteins; 4.69×10¹³ particles) ofconcentrated supernatant of a cell culture (FIG. 7A). The viral particlepeak collected after chromatography (20 ml; 5.6 mg of proteins)contained 4.28×10¹³ particles, which indicates a particle yield of 91%and a purification factor of 32. After this purification step, thefraction collected had (FIGS. 7B-D) a purity greater than 99% as viralparticles during the chromatographic analysis under the conditionsdescribed in Example 5.2 and an absorbance ratio of 1.29. Theelectrophoretic analysis of the adenoviral fraction purified bychromatography carried out under the conditions described in Example 5.2showed that this preparation had a purity level at least equal to thatof the preparation conventionally obtained by ultracentrifugation, andthat it is free of contaminating proteins or of contaminating nucleicacids.

[0179] 5.4. Purification of Intracellular Adenovirus by Strong AnionExchange.

[0180] This example illustrates how an adenovirus comprising in itsgenome a heterologous nucleic acid sequence may be directly purified ina single ion-exchange chromatography step has from a lysate ofencapsidation cells producing the said virus.

[0181] In a typical purification experiment, an adenovirus comprising inits genome a heterologous nucleic acid sequence encoding the β-galprotein is purified by chromatographing in the system described inExample 5.1 (FineLine Pilot 35 column, Pharmacia, 100 ml of Source 15Qresin), 450 ml (that is to say 2.5×10¹⁴ particles) of concentratedlysate of a cell culture harvested 3 days post-infection by chemicallysis (1% Tween-20). The viral particle peak collected afterchromatography (110 ml) contained 2.15×10¹⁴ particles, which indicates aparticle yield of 86%. After this purification step, the fractioncollected had a viral particle purity greater than 98% during thechromatographic analysis under the conditions described in 5.2.Electrophoretic analysis of the adenoviral fraction purified bychromatography carried out under the conditions described in Example 5.2showed that this preparation had a level of purity at least equal hasthat of a preparation conventionally obtained by ultracentrifugation,and that it lacks contaminating proteins or contaminating nucleic acids.

[0182] 5.5. Purification of the Virus by Ultrafiltration andIon-Exchange Chromatography on Various Columns.

[0183] This example illustrates how the adenovirus contained in theconcentrate may be directly purified and in a single ion-exchangechromatography step using a gel different from the Source 15 Q support,while working on the same separation principle, the anion exchange byinteraction with the quaternary amine groups of the matrix.

[0184] In a typical experiment for purification of the adenovirus,various recombinant adenoviruses (encoding βGal, apolipoprotein AI andTK) were purified by chromatography on a Source Q30 gel column followingthe protocol described in Example 5.1. The results obtained show thatthe Source Q30 gel makes it possible to obtain viral preparations of apurity of the order of 85%, with a yield of between 70 and 100%. Inaddition, the results obtained show that Q30 possesses, for thepurification of adenovirus, an efficiency (expressed by the number oftheoretical plates) of 1000 and a (maximum quantity of virus which maybe chromatographed without the peaks becoming altered) of 0.5 to 1×10¹²vp per ml. These results show that the Source Q30 gel may therefore besuitable for the purification of recombinant adenoviruses, even thoughits properties remain inferior to those of Source Q15 (purity of theorder of 99%, efficiency of the order of 8000 and capacity of the orderof 2.5 to 5×10¹² vp per ml).

[0185] In another typical adenovirus purification experiment, a β-Galadenovirus is purified by chromatography on a MonoQ HR 5/5 column usingthe procedure described in Example 5.1. The chromatographic imagecorresponding to the ultrafiltration retentate and to the purified viralpreparation thus obtained is illustrated in FIG. 8.

[0186] In another typical adenovirus purification experiment, a β-Galadenovirus is purified by chromatography on a Poros HQ/M columnaccording to the procedure described in Example 5.1. The chromatographicimage corresponding to the ultrafiltration retentate and to the purifiedviral preparation thus obtained is illustrated in FIG. 9.

Example 6 Purification of the Virus by Ultrafiltration and GelPermeation

[0187] This example illustrates how the adenovirus contained in theconcentrate (ultrafiltration retentate) may be purified directly by gelpermeation chromatography, with very high yields.

[0188] 6.1. Procedure

[0189] 200 μl of the ultrafiltration retentate obtained in Example 4(that is to say 1.3 mg of proteins) are injected into an HR 10/30 column(Pharmacia) filled with Sephacryl S-1000SF (Pharmacia) equilibrated forexample in PBS buffer, pH 7.2 containing 150 mM NaCl (buffer C). Thespecies are fractionated and eluted with buffer C at a flow rate of 0.5ml/min and detected at the outlet of the column by UV at 260 nm.Alternatively, it is possible to use, under the same operatingconditions, a column filled with Sephacryl S-2000, which allows a betterresolution than the column Sephacryl S-1000HR for particles of 100 nm to1000 nm.

[0190] The resolution of the two gel permeation chromatographic systemsdescribed above may be advantageously enhanced by chromatographing theultrafiltration supernatant (200 μl) on a system of 2 HR 10/30 columns(Pharmacia) coupled in series (Sephacryl S-1000HR or S-2000 columnfollowed by a Superdex 200 HR column) equilibrated in the buffer C. Thespecies are eluted with the buffer C at a flow rate of 0.5 ml/min anddetected by UV at 260 nm. In this system, the viral particle peak isvery clearly better separated from the lower-molecular weight speciesthan in the system comprising a Sephacryl S-1000 HR or Sephacryl S-2000column alone.

[0191] In a representative experiment, an ultrafiltration retentate (200μl, 1.3 mg of proteins) was chromatographed on a system of 2 SephacrylS-1000HR-Superdex 200 HR 10/30 columns (FIG. 10). The chromatographicpeak containing the viral particles was collected. Its retention timecoincides with the retention time obtained with a preparation of viralparticles purified by ultracentrifugation. The viral particle peakcollected after chromatography (7 ml) contained 28 μg of proteins and3.5×10⁹ PFU. Its analysis by analytical ion-exchange chromatographyunder the conditions described in Example 5.2 shows the presence of acontaminating peak which is more strongly retained on the analyticalcolumn, whose surface area represents about 25% of the surface area ofthe viral peak. Its 260 nm/280 nm absorbance ratio which has a value of1.86 indicates that this contaminating peak corresponds to nucleicacids. The viral particles were therefore purified about 50 fold (interms of quantity of proteins) and the purification yield is 85% as PFU.

[0192] Alternatively, it is possible to chromatograph the preparationsof viral particles (ultrafiltrates or fractions at the outlet of ananion-exchange chromatography) on a TSK G6000 PW column (7.5×300 mm;TosoHaas) equilibrated in the buffer C. The species are eluted with thebuffer C at a flow rate of 0.5 ml/min and detected in UV at 260 nm.Likewise, it may be advantageous to increase the resolution of thechromatographic system, in particular to increase the separation of theviral particle peak from the lower-molecular weight species, bychromatographing the ultrafiltration supernatant (50 to 200 μl) on asystem of 2 columns coupled in series [TSK G6000 PW column (7.5×300 mm)followed by a Superdex 200 HR column] equilibrated in the buffer C. Thespecies are eluted with the buffer C at a flow rate of 0.5 ml/min anddetected in UV at 260 nm.

Example 7 Purification of the Virus by Ultrafiltration, Ion Exchange andGel Permeation

[0193] The viral particle fraction derived from the anion-exchangechromatography (Example 5) may be advantageously chromatographed in oneof the gel permeation chromatographic systems described above, forexample with the aim of further enhancing the level of purity of theviral particles, but also mainly with the aim of packaging the viralparticles in a medium compatible with or adapted to subsequent uses ofthe viral preparation (injection, . . . )

1 8 1 25 DNA Adenovirus 1 taattacctg ggcggcgagc acgat 25 2 25 DNAAdenovirus 2 accttggatg ggaccgctgg gaaca 25 3 25 DNA Adenovirus 3tttttgatgc gtttcttacc tctgg 25 4 25 DNA Adenovirus 4 cagacagcgatgcggaagag agtga 25 5 25 DNA Adenovirus 5 tgttcccagc ggtcccatcc aaggt 256 25 DNA Adenovirus 6 aaggacaagc agccgaagta gaaga 25 7 25 DNA Adenovirus7 ggatgatatg gttggacgct ggaag 25 8 25 DNA Adenovirus 8 agggcggatgcgacgacact gactt 25

1. Process for the production of recombinant adenoviruses, characterizedin that the viral DNA is introduced into a culture of encapsidationcells and the viruses produced are harvested following release into thesupernatant.
 2. Process according to claim 1, characterized in that theharvesting is performed when at least 50% of the viruses have beenreleased into the supernatant.
 3. Process according to claim 1,characterized in that the harvesting is performed when at least 70% ofthe viruses have been released into the supernatant.
 4. Processaccording to claim 1, characterized in that the harvesting is performedwhen at least 90% of the viruses have been released into thesupernatant.
 5. Process according to claim 1, characterized in that theviruses are harvested by ultrafiltration.
 6. Process according to claim5, characterized in that the ultrafiltration is a tangentialultrafiltration.
 7. Process according to claim 5 or 6, characterized inthat the ultrafiltration is performed on a membrane having a cut-off ofless than 1000 kDa.
 8. Process according to claim 1, characterized inthat the viruses are harvested by anion-exchange chromatography. 9.Process according to claim 8, characterized in that the anion-exchangechromatography is a strong anion-exchange chromatography.
 10. Processaccording to claim 9, characterized in that the strong anion-exchangechromatography is performed on a support chosen from the resins SourceQ, Mono Q, Q Sepharose, Poros HQ and Poros QE, resins of the FractogelTMAE type and Toyopearl Super Q.
 11. Process according to claim 1,characterized in that the viruses are harvested by gel permeationchromatography.
 12. Process according to claim 11, characterized in thatthe gel permeation chromatography is performed on a support chosen fromthe gels Sephacryl S-500 HR, Sephacryl S-1000 SF, Sephacryl S-1000 HR,Sephacryl S-2000, Superdex 200 HR, Sepharose 2B, 4B or 6B and TSK G6000PW.
 13. Process according to claim 1, characterized in that the virusesare harvested by ultrafiltration followed by an anion-exchangechromatography.
 14. Process according to claim 13, characterized in thatthe viruses are harvested by ultrafiltration followed by ananion-exchange chromatography and then a gel permeation chromatography.15. Process according to one of the preceding claims, characterized inthat the encapsidation cell is a cell which transcomplements theadenovirus E1 function.
 16. Process according to claim 15, characterizedin that the encapsidation cell is a cell which transcomplements theadenovirus E1 and E4 functions.
 17. Process according to claim 15,characterized in that the encapsidation cell is a cell whichtranscomplements the adenovirus E1 and E2a functions.
 18. Processaccording to one of claims 15 to 17, characterized in that the cell isan embryonic human kidney cell, a human retinoblast or a cell from ahuman carcinoma.
 19. Process for the purification of recombinantadenoviruses from a biological medium characterized in that it comprisesa step of purification by strong anion-exchange chromatography. 20.Process according to claim 19, characterized in that the biologicalmedium is a supernatant of encapsidation cells producing the said virus.21. Process according to claim 19, characterized in that the biologicalmedium is a lysate of encapsidation cells producing the said virus. 22.Process according to claim 19, characterized in that the biologicalmedium is a prepurified solution of the said virus.
 23. Processaccording to claim 19, characterized in that the chromatography iscarried out on a support functionalized with a quaternary amine. 24.Process according to claim 23, characterized in that the support ischosen from agarose, dextran, acrylamide, silica,poly[styrene-divinylbenzene], ethyleneglycol-methacrylate copolymer,alone or as a mixture.
 25. Process according to claim 24, characterizedin that the chromatography is performed on a resin Source Q, Mono Q, QSepharose, Poros HQ, Poros QE, Fractogel TMAE or Toyopearl Super Q. 26.Process according to claim 25, characterized in that the chromatographyis performed on a Source Q resin, preferably Source Q15.
 27. Processaccording to claim 19, characterized in that it comprises a preliminaryultrafiltration step.
 28. Process according to claim 27, characterizedin that the ultrafiltration is a tangential ultrafiltration on amembrane having a cut-off of between 300 and 500 kDa.
 29. Purified viralpreparation obtained according to the process of claim 1 or
 19. 30.Pharmaceutical composition comprising a viral preparation according toclaim 29 and a pharmaceutically acceptable vehicle.
 31. Use ofiodixanol, 5,5′-[(2-hydroxy-1,3-propanediyl)bis(acetylimino)]bis[N,N′-bis(2,3-dihydroxypropyl)-2,4,6-triiodo-1,3-benzenecarboxamide]for the purification of adenoviruses.
 32. Process for the purificationof adenoviruses from a biological medium comprising a first step ofultracentrifugation, a second step of dilution or dialysis, and a thirdstep of anion-exchange chromatography.